Project Summary Iron is an essential nutrient for humans, yet excess iron is toxic. As such, iron overload and iron deficiency result in severe homeostatic perturbations. Iron overload is most frequently associated with hereditary hemochromatosis (HH), which afflicts ~1:250 adults in the U.S. Tissue iron accumulation in patients with HH leads to arthritis, osteoporosis, liver damage and cancer, cardiomyopathy, diabetes mellitus, and impotence. HH results from impaired production of the iron-regulatory hormone hepcidin (HEPC) or as a result of mutations in the HAMP gene (encoding HEPC). HEPC limits intestinal iron absorption. Moreover, reduced HEPC synthesis underlies the iron loading that typifies disorders of ineffective erythropoiesis (e.g. ?- thalassemia intermedia [?TI]). In HH and ?TI, intestinal iron absorption is thus excessive. This leads to pathological iron overload since humans cannot excrete excess iron. Regulation of intestinal iron absorption is thus critical to properly control body iron levels. Dietary iron exists primarily as inorganic (or nonheme) iron. Ferric (Fe3+) nonheme iron is first reduced to Fe2+, imported into duodenal enterocytes by divalent metal-ion transporter 1 (DMT1), exported by ferroportin 1 (FPN1) and oxidized for binding to transferrin. DMT1 is the primary intestinal iron importer under basal conditions, but the relative contribution of DMT1 to iron accumulation in HH and ?TI is unknown. In Aim 1, we will thus test the hypothesis that DMT1 is required for iron loading in mouse models of HH (HEPC KO) and ?TI (Hbbd3th; with a mutated ? major globin [Hbb- b1]) gene. We will thus generate HEPC and Hbb-b1 KO mice that are also lacking intestinal DMT1. We further hypothesize that decreasing DMT1 expression will prevent iron loading in HH and ?TI. Accordingly, we have developed ginger nanoparticle-derived lipid vectors (GNLVs) which can deliver functional DMT1 siRNA to the mouse duodenum in vivo (~40% reduction in DMT1 expression). In Aim 2, this GNLV delivery system will be tested for its ability to prevent iron loading in rodent models of HH and ?TI. Furthermore, iron deficiency (ID) is also common in the U.S., afflicting ~8 million young women, and 700,000 infants. ID frequently occurs when absorption of dietary iron does not meet the body?s demand. ID most commonly occurs as a consequence of rapid growth, pregnancy, menstrual blood loss, malabsorptive disorders, gastric bypass surgery and chronic inflammation. ID symptoms include anemia, impaired cognition, decreased immune response, and fatigue. During ID, DMT1 increases Cu transport into duodenal enterocytes and emerging data demonstrates that copper is critical to support iron repletion during states of deficiency. The mechanism that transforms DMT1 into a copper transporter is, however, unknown. Aim 3 will thus test the hypothesis that the DMT1 protein is post-translationally modified during iron deficiency, allowing Cu transport. Plausible alternative hypotheses may also be considered. Overall, this DMT1-focused investigation is likely to potentiate the development of novel therapeutic and nutritional approaches to modulate intestinal iron absorption in at-risk individuals.